Background: Friedreich ataxia (FRDA), the most common form of recessive ataxia, is due to reduced levels of frataxin, a highly conserved mitochondrial iron-chaperone involved in iron-sulfur cluster (ISC) biogenesis. Most patients are homozygous for a (GAA)(n) expansion within the first intron of the frataxin gene. A few patients, either with typical or atypical clinical presentation, are compound heterozygous for the GAA expansion and a micromutation.

Methodology: We have developed a new strategy to generate murine cellular models for FRDA: cell lines carrying a frataxin conditional allele were used in combination with an EGFP-Cre recombinase to create murine cellular models depleted for endogenous frataxin and expressing missense-mutated human frataxin. We showed that complete absence of murine frataxin in fibroblasts inhibits cell division and leads to cell death. This lethal phenotype was rescued through transgenic expression of human wild type as well as mutant (hFXN(G130V) and hFXN(I154F)) frataxin. Interestingly, cells expressing the mutated frataxin presented a FRDA-like biochemical phenotype. Though both mutations affected mitochondrial ISC enzymes activities and mitochondria ultrastructure, the hFXN(I154F) mutant presented a more severe phenotype with affected cytosolic and nuclear ISC enzyme activities, mitochondrial iron accumulation and an increased sensitivity to oxidative stress. The differential phenotype correlates with disease severity observed in FRDA patients.

Conclusions: These new cellular models, which are the first to spontaneously reproduce all the biochemical phenotypes associated with FRDA, are important tools to gain new insights into the in vivo consequences of pathological missense mutations as well as for large-scale pharmacological screening aimed at compensating frataxin deficiency.

Mentions:
All isolated clones were viable and proliferated over multiple passages in classical culture conditions. However, compared to the clones expressing wild type hFXN, the hFXNI154F expressing clones repeatedly showed a growth defect, taking a longer time to reach confluence. This growth defect was particularly noticeable after strong dilution of the cell line. Although consistent, the growth defect was not sufficiently severe to cause a significant difference over a 4-day proliferation curve (data not shown). Interestingly, the hFXNG130V expressing clones did not show any growth defect. Furthermore, while the clones expressing the wild type hFXN presented normal fibroblast morphology (Fig. 3, line hFXN-1D12), the hFXNG130V expressing clones displayed a slightly altered morphology with smaller or less spread out cells (Fig. 3, line hFXNG130V-G4). In correlation with the growth defect, the hFXNI154F expressing clones displayed a very altered morphology, with numerous small grainy rounded cells with a retracted cytoplasm and some elongated spindle-shaped cells (Fig. 3, lines hFXNI154F-1D3 and hFXNI154F-2C1). Finally, gigantic cells reminiscent of senescence features were also observed in the three hFXNI154F expressing clones. These cells were spread out and exhibited fractionated nucleus and cytosolic vacuoles (Fig. 3, line I154F-1D3).

Mentions:
All isolated clones were viable and proliferated over multiple passages in classical culture conditions. However, compared to the clones expressing wild type hFXN, the hFXNI154F expressing clones repeatedly showed a growth defect, taking a longer time to reach confluence. This growth defect was particularly noticeable after strong dilution of the cell line. Although consistent, the growth defect was not sufficiently severe to cause a significant difference over a 4-day proliferation curve (data not shown). Interestingly, the hFXNG130V expressing clones did not show any growth defect. Furthermore, while the clones expressing the wild type hFXN presented normal fibroblast morphology (Fig. 3, line hFXN-1D12), the hFXNG130V expressing clones displayed a slightly altered morphology with smaller or less spread out cells (Fig. 3, line hFXNG130V-G4). In correlation with the growth defect, the hFXNI154F expressing clones displayed a very altered morphology, with numerous small grainy rounded cells with a retracted cytoplasm and some elongated spindle-shaped cells (Fig. 3, lines hFXNI154F-1D3 and hFXNI154F-2C1). Finally, gigantic cells reminiscent of senescence features were also observed in the three hFXNI154F expressing clones. These cells were spread out and exhibited fractionated nucleus and cytosolic vacuoles (Fig. 3, line I154F-1D3).

Background: Friedreich ataxia (FRDA), the most common form of recessive ataxia, is due to reduced levels of frataxin, a highly conserved mitochondrial iron-chaperone involved in iron-sulfur cluster (ISC) biogenesis. Most patients are homozygous for a (GAA)(n) expansion within the first intron of the frataxin gene. A few patients, either with typical or atypical clinical presentation, are compound heterozygous for the GAA expansion and a micromutation.

Methodology: We have developed a new strategy to generate murine cellular models for FRDA: cell lines carrying a frataxin conditional allele were used in combination with an EGFP-Cre recombinase to create murine cellular models depleted for endogenous frataxin and expressing missense-mutated human frataxin. We showed that complete absence of murine frataxin in fibroblasts inhibits cell division and leads to cell death. This lethal phenotype was rescued through transgenic expression of human wild type as well as mutant (hFXN(G130V) and hFXN(I154F)) frataxin. Interestingly, cells expressing the mutated frataxin presented a FRDA-like biochemical phenotype. Though both mutations affected mitochondrial ISC enzymes activities and mitochondria ultrastructure, the hFXN(I154F) mutant presented a more severe phenotype with affected cytosolic and nuclear ISC enzyme activities, mitochondrial iron accumulation and an increased sensitivity to oxidative stress. The differential phenotype correlates with disease severity observed in FRDA patients.

Conclusions: These new cellular models, which are the first to spontaneously reproduce all the biochemical phenotypes associated with FRDA, are important tools to gain new insights into the in vivo consequences of pathological missense mutations as well as for large-scale pharmacological screening aimed at compensating frataxin deficiency.